REVIEW published: 06 April 2017 doi: 10.3389/fpls.2017.00494

Non-canonical Translation in Plant RNA Viruses

Manuel Miras 1, W. Allen Miller 2, Verónica Truniger 1 and Miguel A. Aranda 1*

1 Centro de Edafología y Biología Aplicada del Segura - CSIC, Murcia, Spain, 2 Department of Plant Pathology and Microbiology, Iowa State University, Ames, IA, USA

Viral protein synthesis is completely dependent upon the host cell’s translational machinery. Canonical translation of host mRNAs depends on structural elements such as the 5′ cap structure and/or the 3′ poly(A) tail of the mRNAs. Although many viral mRNAs are devoid of one or both of these structures, they can still translate efficiently using non-canonical mechanisms. Here, we review the tools utilized by positive-sense single-stranded (+ss) RNA plant viruses to initiate non-canonical translation, focusing on cis-acting sequences present in viral mRNAs. We highlight how these elements may interact with host translation factors and speculate on their contribution for achieving translational control. We also describe other translation strategies used by plant viruses to optimize the usage of the coding capacity of their very compact genomes, including leaky scanning initiation, ribosomal frameshifting and stop-codon readthrough. Finally, future research perspectives on the unusual translational strategies of +ssRNA viruses Edited by: are discussed, including parallelisms between viral and host mRNAs mechanisms of Mar Castellano, translation, particularly for host mRNAs which are translated under stress conditions. INIA, Spain Reviewed by: Keywords: non-canonical translation, RNA structure and function, translation enhancers, translational recoding, ′ Lyuba A. Ryabova, protein synthesis, IRES, 3 -CITE Institute of Plant Molecular Biology, France Aurelie Rakotondrafara, INTRODUCTION University of Wisconsin-Madison, USA *Correspondence: Viruses usurp the metabolism of the host cell in their own benefit. Viral mRNA translation is a Miguel A. Aranda paradigmatic illustration of this, as the hallmark of viruses is that their genomes do not code for [email protected] a protein synthesis apparatus. Thus, viruses have evolved many subtle ways to use and control the translational machinery of their hosts (Jiang and Laliberté, 2011; Echevarría-Zomeño et al., 2013; Specialty section: Walsh et al., 2013), and in fact the host range of a given virus may be determined by its ability to This article was submitted to efficiently translate viral mRNAs using host translation factors, as we have shown recently for a Plant Physiology, plant virus (Truniger et al., 2008; Nieto et al., 2011; Miras et al., 2016). From a strategic point of a section of the journal view, understanding how viruses translate their own proteins may significantly contribute to the Frontiers in Plant Science identification of therapeutic (Robert et al., 2006; Cencic et al., 2011) or breeding targets (Nicaise Received: 28 February 2017 et al., 2003; Gao et al., 2004; Ruffel et al., 2005; Stein et al., 2005; Nieto et al., 2006; Naderpour Accepted: 21 March 2017 et al., 2010). Also, understanding the peculiarities of viral mRNA translation can provide important Published: 06 April 2017 biotechnological tools for protein overexpression (Sainsbury and Lomonossoff, 2014; Lomonossoff Citation: and D’Aoust, 2016), given the very efficient translation of some viral mRNAs in diverse conditions. Miras M, Miller WA, Truniger V and From a fundamental point of view, viral mRNAs constitute powerful probes to uncover the varied Aranda MA (2017) Non-canonical Translation in Plant RNA Viruses. and fascinating mechanisms of protein translation and their control. In this review, we describe Front. Plant Sci. 8:494. current knowledge on the mechanisms used by positive-sense single-stranded (+ss) RNA plant doi: 10.3389/fpls.2017.00494 viruses to initiate translation, focusing on cis-acting sequences present in viral mRNAs. We also

Frontiers in Plant Science | www.frontiersin.org 1 April 2017 | Volume 8 | Article 494 Miras et al. Non-canonical Translation in Plant RNA Viruses describe other protein translation strategies used by plant viruses Once the mRNA is circularized, the 43S PIC in its open to optimize the usage of the coding capacity of their very conformation is able to bind to the mRNA near its 5′ end. compact genomes, including leaky scanning initiation, ribosomal The exact mechanistic details are unknown, but eIF3 and eIF4G frameshifting and stop-codon readthrough. appear to facilitate this step (Aitken and Lorsch, 2012). The 43S PIC searches for the mRNA start codon, scanning downstream of the leader sequence resulting in the entry of the 5′ proximal CANONICAL TRANSLATION OF start codon into the 40S subunit P-site (Kozak, 2002). Start codon EUKARYOTIC mRNAs selection requires cooperation between the scanning ribosome and eIF1, eIF2, and eIF5, forming the 48S preinitiation complex To understand the mechanisms of non-canonical translation of (Pestova and Kolupaeva, 2002). Once the start codon enters viral mRNAs, we first review briefly how canonical eukaryotic the P-site, the 60S subunit joins, with the release of eIF2, eIF1, mRNA translation proceeds. Most eukaryotic mRNAs are and eIF5 and the association with eIF5B-GTP (Pestova et al., appended at the 5′ end with a m7G(5′)ppp(5′)N cap structure, 2001). With the formation of the resulting 80S complex, the and a poly(A) tail at the 3′ end, which are critical cis-acting GTP molecule associated with eIF5B is hydrolyzed and released elements during canonical translation. Traditionally, translation (Pestova et al., 2001). is divided into four distinct steps: initiation, elongation, Translation continues with the elongation phase, where the termination and ribosomal recycling. Translation initiation is polypeptide is formed. In the elongation stage, entering amino the rate limiting and most highly regulated step (reviewed in acyl-tRNAs (aa-tRNA) bind to the A-site through the second Aitken and Lorsch, 2012) and begins with the formation of the codon of the mRNA (Lewin, 2008). After the aa-tRNA is located 43S preinitiation complex (PIC). PIC is composed of the ternary at the A-site, the peptidyl-tRNA is relocated from the P-site to complex (TC) eIF2-Met-tRNA-GTP bound to the 40S ribosome the A-site. Once the peptide bond is formed, the translocation subunit through the P-site and the eukaryotic initiation factors step occurs when the ribosome moves in a 3′ direction along the (eIFs) eIF3, eIF5, eIF1A, and eIF1 (Sonenberg and Hinnebusch, mRNA, placing a new codon at an empty A-site while the new 2009). EIF3, which is a large thirteen-subunit complex (Sun et al., peptidyl-tRNA is moved to the P-site and the deacylated tRNA 2011; Browning and Bailey-Serres, 2015; Smith et al., 2016), in the E-site is ready to exit the ribosome (Julián et al., 2008; interacts with eIF2 via its subunit eIF3a and indirectly via eIF5 Rodnina and Wintermeyer, 2009). After the nascent polypeptide bridging these two factors (Valášek et al., 2002; Jivotovskaya et al., has been released, ribosomes remain bound to the mRNA and 2006). Interestingly, the eIF3d subunit can act as a cap-binding tRNA. It is only during the ribosomal recycling phase when protein and is required for specialized cap-dependent translation the ribosome subunit dissociation occurs leaving them free to (Lee et al., 2016). bind new mRNAs (Pisareva et al., 2011; Dever and Green, In parallel to PIC formation, recognition of the mRNA is 2012). facilitated through binding of the cap-binding protein eIF4E to ′ the 5’ cap and the poly(A)-binding protein (PABP) to the 3 NON-CANONICAL TRANSLATION poly(A) tail (Pestova et al., 2001). EIF4G interacts with eIF4E through its highly conserved canonical binding domain and INITIATION OF VIRAL mRNAs forms, together with the helicase eIF4A, the eIF4F complex. Very Mechanisms of non-canonical translation initiation include recently, a second eIF4E-binding domain has been described those that function independently of a 5′ cap or/and a poly(A) in eIF4G, suggesting a bipartite eIF4E-eIF4G binding mode tail. These can be mediated by stimulators present in cis in the for higher eukaryotes (Grüner et al., 2016). EIF4G can also 5′-UTR, for example internal ribosome entry sites (IRESes) or recruit other factors, including eIF3 and PABPs through direct genome-linked viral proteins (VPgs), in the 3′-UTR, for example protein-protein interactions. It is thought that the eIF4G- cap-independent translation elements (3′-CITE) or tRNA-like PABP interaction promotes the circularization of the message structures (TLS), and also in intergenic regions, for example enhancing translation efficiency (Gray et al., 2000; Paek et al., intergenic IRESes (Table 1). 2015). This model is supported by biochemical data and by atomic force microscopy studies that confirm the interactions Enhancers Located in the 5′-UTR: Internal and the circularization of the mRNA (Wells et al., 1998; Kahvejian et al., 2001). However, there is increasing evidence Ribosome Entry Sites and VPgs in that circularization may vary in importance for stimulation of Potyviridae translation among different organisms (i.e., yeast) and cells types. The family Potyviridae is the largest among plant viruses with For example, the eIF4G-PABP interaction is not required for RNA genomes. The potyviral genome acts as mRNA and codes wild-type cell growth in yeast and mammals (Hinton et al., 2007; for a single polyprotein which is cleaved by viral proteases Park et al., 2011). Similarly, it was observed by cryo-EM that rendering 10 final functional proteins (Revers and García, the formation of circular polyribosomes was independent of the 2015). Potyviral RNAs resemble those of the animal-infecting cap structure and poly(A) tail (Madin et al., 2004; Afonina et al., picornaviruses: they possess a small viral protein covalently 2014). These results suggest alternative mechanisms for mRNA bound to their 5′ ends (VPg), instead of a 5′ cap structure, and circularization that may mimic the strategies used by +ssRNA they are polyadenlylated at their 3′ ends (Adams et al., 2005). viruses detailed in this review. However, VPgs in different virus families differ greatly in size

Frontiers in Plant Science | www.frontiersin.org 2 April 2017 | Volume 8 | Article 494 Miras et al. Non-canonical Translation in Plant RNA Viruses References 2006 2011 2004 Zeenko and Gallie, 2005; Ray et al., Yang et al., 1997 Basso et al., 1994; Yang et al.,Roberts 2009 et al., 2015, 2017 Karetnikov and Lehto, 2007 Jaag et al., 2003 Koh et al., 2003 Fernández-Miragall and Hernández, May et al., 2017 Dorokhov et al., 2002 Matsuda et al., 2004; Colussi et al.,Barends 2015 et al., 2004 Neeleman et al., 2004; Krab et al., 2005 Meulewaeter et al., 1998; Gazo et al., Blanco-Pérez et al., 2016 Truniger et al., 2008; Miras et al.,Nicholson 2016 et al., 2010 Miras et al., 2014 Treder et al., 2008; Sharma et al.,Wang 2015 et al., 2010 Wang et al., 2010 Shen and Miller, 2004 Wang et al., 2010 Fabian and White, 2004 Nicholson et al., 2013 Wang et al., 2009b, 2011 Batten et al., 2006 Chattopadhyay et al., 2011 Stupina et al., 2008; Zuo et al.,Gao 2010 et al., 2013, 2014 Karetnikov et al., 2006        complementarity eIF4F/iso4F eEF1a/ 40S eIF4E independent eIF4E 60S 40S/60S eIF4G eIF4G RPS6 eIF4G/40S eIF4G eIF4G eIF4G/eIFiso4G eIF4F/eIFiso4F eIF4F eIF4F CP/eIF4G/iso4G a a a a a a b b b b b a a a b a a b a a a b b a a a a a RNA secondary structure eIFs/Other 18S RNA None Two stem-loops Stem-loop Bulge stem-loop Bulge stem-loop Bulge stem-loop Stem-loop Two stem-loops -CITE ′ Type of 3 -UTR TED Long stem-loop -UTR tRNA-like -UTR Two pseudoknots -UTR Six stem-loops ′ ′ ′ ′ localization structures Genome ′ GpppN/TLS 3 GpppN/TLS GpppN/TLS GpppN/-OH 3 /3 pppN/-OH 3 pppN/-OH pppN/-OH pppN/-OH pppN/-OHpppN/-OHpppN/-OHpppN/-OHpppN/-OHpppN/-OH TEDpppN/-OH ISSpppN/-OH ISS Long stem-loop pppN/-OH CXTE Stem-loop pppN/-OH Stem-loop pppN/-OH Two helices protruding central hub pppN/-OH BTEpppN/-OH BTEpppN/-OH BTE Basal helix plus 5 helices YSS Basal helix plus 2 helices YSS Basal helix plus 2 helices PTE Three helices PTE Three helices PTE Pseudoknot eIF4G TSS Pseudoknot TSS Pseudoknot tRNA-like tRNA-like pppN/-OH Intergenic region Bulge stem-loop pppN/-OHpppN/-OH BTE BTE Basal helix plus 3 helices Complex BTE structure 7 7 7 7 ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ ′ VPg/poly(A)VPg/poly(A) 5 VPg/poly(A) 5 m m m 5 5 VPg/poly(A) – Pseudoknot 5 5 5 5 m 5 5 5 5 5 5 5 5 5 5 5 5 5 5 VPg/poly(A) VPg/poly(A) Tombusviridae Potyviridae Potyviridae Secoviridae Luteoviridae Tombusviridae Tombusviridae Tombusviridae Virgaviridae Tymoviridae Bromoviridae Bromoviridae Tombusviridae Tombusviridae Tombusviridae Tombusviridae Luteoviridae Luteoviridae Tombusviridae Tombusviridae Tombusviridae Tombusviridae Tombusviridae Tombusviridae Tombusviridae Tombusviridae Tombusviridae Tombusviridae Secoviridae Potyviridae Potyviridae PVY TuMV TriMV BRV HCRSV PFBV TCV crTMV BMV PLPV MNSV MNeSV MNSV-N BYDV RSDaV RCNMV TNV-D TBTV TBSV CIRV PEMV2 PMV SCV TCV PEMV2 BRV CITE STNV ′ Predicted RNA secondary structure. Solution probed RNA secondary structure. TABLE 1 | Translation enhancers known in RNA plant viruses. Type VirusIRES Family TEV 5 IRES PRLV TLS TYMV CPB AMV 3 a b

Frontiers in Plant Science | www.frontiersin.org 3 April 2017 | Volume 8 | Article 494 Miras et al. Non-canonical Translation in Plant RNA Viruses and function. The well-characterized VPg of Poliovirus (genus translation of an ORF in a dicistronic vector (Levis and Astier- Enterovirus, family Picornaviridae) is only 22 amino acids (aa) Manifacier, 1993), and IRES mapping showed that a 55 nt 3′ long, while that of potyviruses consists of around 192 aa. terminal region was fundamental for translation enhancement Early studies using the model potyvirus Tobacco etch in tobacco protoplasts (Yang et al., 1997). The 131-nt long 5′ virus (TEV, genus Potyvirus, family Potyviridae) showed that leader of TuMV conferred translational activity when placed its 5′-UTR contains a sequence that was able to enhance upstream of a GUS reporter gene flanked at its 5′ end by a translation 8- to 21-fold in tobacco protoplasts (Carrington 33 nt vector-sequence (Basso et al., 1994); this RNA was able and Freed, 1990). Deletion studies identified two regions in to promote translation in vitro to a similar level as capped the TEV 5′-UTR including nucleotides 26-85 and 66-118 which mRNAs inhibiting cap-dependent translation when added in were able to stimulate translation 10-fold with respect to a trans (Basso et al., 1994). The study from Yang et al. (2009) capped RNA control (Zeenko and Gallie, 2005); these regions demonstrated that the TuMV RNA requires the ribosomal were consequently named cap-independent regulatory elements protein RPS6 for accumulation in Nicotiana benthamiana, and (CIRE) 1 and 2 (Zeenko and Gallie, 2005). The TEV CIREs RPS6 is up-regulated under TuMV infection in Arabidopsis promoted translation of a second ORF when placed in a thaliana. The silencing of RPS6 abolished TuMV infection and dicistronic reporter construct, suggesting that they were able also that of the non-related Tomato bushy stunt virus (TBSV; to promote internal initiation like IRESes (Niepel and Gallie, genus Tombusvirus, family Tombusviridae) (Yang et al., 2009). 1999). However, the addition of a stem loop structure upstream The TBSV viral RNA is uncapped and not polyadenylated, of CIRE-1 and CIRE-2 in its natural 5′ end context reduced having no VPg. The RPS6 protein is related to other ribosomal translation 30 and 70%, respectively, suggesting that the TEV proteins implicated in picornaviral and alphaviral infection and leader might require an accessible 5′ end for ribosomal scanning indispensable for Hepatitis C virus (HCV, genus Hepacivirus, (Niepel and Gallie, 1999). The TEV CIRE-1 folds into an AU- family Flaviviridae) replication (Cherry et al., 2005; Montgomery rich pseudoknot structure (PK1, nucleotides 38–75) which is et al., 2006; Huang et al., 2012). essential for cap-independent translation. Interestingly, one loop It should be noted that the above reported IRESes of of PK1 is complementary to a conserved region of the 18S potyviruses may not be as strong as the IRESes of picornaviruses rRNA and mutations in the 7 nt-complementary sequence (61- or HCV, for example. The 5′-UTRs of potyviruses are much UACUUCU-67) were responsible for an approximately 80% shorter than the IRESes of the Picornaviridae, and lack strong decrease in translation compared to wild type (Zeenko and Gallie, structure or conserved sequence, and AUG triplets (Niepel 2005). This type of complementarity also occurs between the 18S and Gallie, 1999; Zeenko and Gallie, 2005). As mentioned rRNA and the sequence 4836-GAUCCU-4841 that belongs to the above, an upstream stem-loop inhibited downstream translation translation enhancer located in the 3′-UTR of Barley yellow dwarf mediated by the IRES, which lends doubt on whether it truly virus (BYDV; genus Luteovirus, family Luteoviridae) (see Section facilitates internal ribosome entry. Moreover, translation directed on CITEs) and the polypyrimidine-rich tracts located in both by the TEV 5′-UTR sequence from the internal position was IRES elements found in Blackcurrant reversion virus (BRV; genus orders of magnitude less efficient than when located at the Nepovirus, family Comoviridae) (Karetnikov and Lehto, 2007; natural 5′ end (Niepel and Gallie, 1999). Also, capped potyviral Sharma et al., 2015), suggesting that these translation elements transcripts containing the 5′-UTR (including the IRES), linked could recruit the 40S ribosomal subunit before loading to the5′ to a reporter gene, translated more efficiently than uncapped end of the mRNA to start the scanning. transcripts (Carrington and Freed, 1990; Khan et al., 2008). These Early experiments using partially eIF4F depleted wheat germ observations support the notion that conventional ribosome extract showed that the TEV 5′-UTR conferred a competitive scanning from the 5′ end is important for efficient translation of advantage over non-viral mRNAs which seemed to be lost potyviral RNAs. when eIF4F was added back to wheat germ extract (Gallie and One singular potyviral 5′-UTR that resembles a true animal Browning, 2001). These results suggest that the TEV genome virus-like IRES, is that of Triticum mosaic virus (TriMV) recruits eIF4F more efficiently than plant mRNAs when the (genus Tritimovirus, Potyviridae). The exceptionally long (739 concentration of this factor is limiting. Further analysis showed nt) 5′-UTR is much longer than that of other potyvirids and that, like for Picornaviridae IRESes, TEV translation is eIF4F- translation initiates at the 13th AUG triplet (Roberts et al., 2015). dependent and that eIF4G binds directly to both, the TEV The minimal region of the TriMV leader for cap-independent 5′ leader and PK1 having a large entropic contribution (Ray translation resides in a 300-nt long sequence forming a secondary et al., 2006). Moreover, the poly(A) tail functions synergistically structure consisting of two long stem-loop-containing bulges.A with the TEV IRES to increase translation (Gallie et al., 1995), hairpin structure at nucleotide positions 469-490 is required for as also shown for animal-infecting picornaviral IRES-mediated cap-independent translation and internal translation initiation, translation (de Quinto et al., 2002; Thoma et al., 2004). and plays a role in its ability to compete with capped RNAs Like that of TEV, the 5′ leaders of Potato virus Y (PVY; (Roberts et al., 2015). A unique feature of the TriMV IRES genus Potyvirus, family Potyviridae), Turnip mosaic virus (TuMV, compared to those of other potyviruses is that it can mediate genus Potyvirus, family Potyviridae), and Triticum mosaic virus translation when a stem-loop structure is added upstream of (TriMV; genus Poacevirus, family Potyviridae) (Table 1) have the 5′ leader, thus its translation is 5′ end independent. The been shown to stimulate cap-independent translation. The 5′- TriMV 5′-UTR interacts with eIF4G or eIFiso4G in vitro, and UTR of PVY also contains an IRES that directs efficient requires eIF4A helicase activity to mediate translation initiation

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(Roberts et al., 2017). These properties are true hallmarks of an (Leen et al., 2016), with this latter domain differing from the IRES. eIF4E-interacting domains in FCV and PSaV VPgs. VPgs vary The VPg covalently attached to the 5′ end of potyviral RNAs widely in sequence, even within a genus, so it would be difficult may contribute directly to translational efficiency by interacting to extrapolate this structural information to potyvirus VPgs. with translation initiation factors (Khan et al., 2008; Miyoshi Instead, to experimentally determine whether the potyvirus VPg et al., 2008). The addition of the TEV VPg together with plays the role of replacing the 5′ cap in translation, it would eIF4F to a depleted wheat germ extract enhanced translation be valuable to determine whether translating potyvirus RNA on of an uncapped TEV RNA reporter (Khan et al., 2008). This polysomes contains a VPg, and the effect of removing this VPg enhancement correlated with an increase in the eIF4F-TEV on potyvirus RNA translation. RNA affinity in the presence of the VPg mediated through Viruses in the family Secoviridae and in the genus Sobemovirus a direct interaction of the VPg with eIF4E. The disruption also have VPgs linked to their genomic RNA. The VPg of of VPg-eIF4E binding abolished stimulation of IRES-mediated the sobemovirus Rice yellow mottle virus has been shown to translation in vitro (Khan et al., 2008). In contrast, TuMV interact with eIFiso4G and this interaction is required for viral VPg binds the isoform of eIF4E, eIFiso4E in vitro and in multiplication, but a role in translation has not been published vivo (Leonard et al., 2004; Khan et al., 2008). PABP increases for this interaction (Hébrard et al., 2010). The role in translation the binding affinity and stabilization of VPg with eIF4F or of secovirids VPgs is poorly understood (Léonard et al., 2002). eIFiso4F in both viruses (Khan et al., 2009; Khan and Goss, 2012). Similarly to the TEV and the TuMV VPg, Potato virus Intergenic Region Enhancers A (PVA, family Potyviridae) VPg binds eIF4E and eIFiso4E IRESes have also been found in internal genomic positions and enhances viral translation in plants (Eskelin et al., 2011). within certain viral genomes (Table 1). For example, the crucifer Silencing of those host factors abolished PVA VPg-mediated strain of Tobacco mosaic virus (crTMV; genus Tobamovirus, stimulation of translation. Ribosomal protein P0 enhanced family Virgaviridae) harbors two IRESes that stimulate the translation synergistically together with VPg and eIFiso4E and its synthesis of the CP and movement protein (MP), 75 and 148- stimulation depended on the PVA 5′-UTR (Hafrén et al., 2013). nucletotides long, respectively (Dorokhov et al., 2002, 2006). Further on, Hafrén et al. (2015) showed that viral HC-Pro and The CP IRES contains a bulged stem-loop structure that is the host RNA binding protein varicose, both components of flanked by two purine-rich repeats that are crucial for IRES potyviral RNA granules, stimulated VPg-promoted translation of activity. To find the minimal purine-rich sequence the authors PVA. reported that 16 consecutive GAAA repeats were sufficient to All of the above mechanisms involve the VPg stimulating provide high IRES activity in plants and human cells (Dorokhov RNA translation in trans, leaving open the question of how the et al., 2002). However, apparently this observation has not been VPg specifically recognizes only the viral RNA. It is unknown repeated in other labs (e.g., Fan et al., 2012). A low level of CP whether the VPg acts in cis when it is covalently attached to translation from genomic RNA of carmoviruses Hibiscus chlorotic the 5′ end, to simply replace the 5′ cap function in recruiting ringspot virus (HCRSV) (Koh et al., 2003; Fernández-Miragall eIF4E and stimulating translation. The much smaller VPg of and Hernández, 2011), Pelargonium flower break virus (PFBV) picornaviruses does not participate in translation, as polysome- (Fernández-Miragall and Hernández, 2011), and Turnip crinkle associated picornaviral RNA lacks the VPg (Nomoto et al., 1977). virus (TCV) (May et al., 2017) has also been reported to be IRES- Instead it primes picornavirus RNA synthesis (Paul et al., 1998). mediated. Like the crTMV IRES, the TCV IRES seems to require It is likely that the VPgs of all viruses also have this latter role, only to be A-rich and lack of structure and its activity is inversely but to our knowledge, priming of RNA synthesis has not been correlated with the size of the RNA. demonstrated for the VPg of any plant virus. Another virus that shares the crTMV polypurine tract in its The potyvirus VPg may functionally resemble the 13–15 IRES sequence is Potato leafroll virus (PLRV; genus Polerovirus, kDa VPg of calici- and noroviruses (Caliciviridae)(Goodfellow, family Luteoviridae). This IRES, which is in a highly unexpected 2011). Like the potyvirus VPg, calicivirus VPg binds eIF4E location, 22 nt downstream of the start codon and within a region (Goodfellow et al., 2005). This interaction is required for of the PLRV RNA genome that is characterized by non-canonical translation of Feline calicivirus (FCV, genus Vesivirus, family translation mechanisms such as −1 ribosomal frameshifting, Caliciviridae) RNA, so the VPg acts as a functional analog of leads to translation of replication-associated protein (Rap1) (Jaag the cap (Goodfellow et al., 2005; Hosmillo et al., 2014; Zhu et al., 2003). The PLRV IRES element, in conjunction with the et al., 2015). In contrast, the VPg on norovirus RNA binds 22 nt spacer sequence, are sufficient to mediate cap-independent and requires eIF4G for translation initiation (Chung et al., translation in vitro but not in vivo (Jaag et al., 2003), which sheds 2014). This difference in factor binding may be associated doubt on its biological relevance. Furthermore, this reported with the different structures of their VPgs. While FCV and IRES function and the resulting translated ORF are not conserved Porcine sapovirus (PSaV, genus Sapovirus, family Caliciviridae) in related poleroviruses. VPgs adopt a compact three-helical bundle structure, Murine Given the unstructured and sequence non-specific nature norovirus (MNV, genus Norovirus, family Caliciviridae) VPg of the IRES RNA in the examples above, which is unlike the has only two helices (Leen et al., 2013; Hwang et al., 2015). much longer, highly structured and powerful mammalian viral The MNV VPg-eIF4G interaction was mapped to the HEAT- and dicistrovirus IRESes, we think these observations should be 1 domain in eIF4G and to the 20 C-terminal residues in VPg interpreted with caution. It may be possible that, due to lack of

Frontiers in Plant Science | www.frontiersin.org 5 April 2017 | Volume 8 | Article 494 Miras et al. Non-canonical Translation in Plant RNA Viruses structure, the RNA is sensitive to nuclease cleavage providing structure of the TYMV TLS. The TLS has a tRNA-like shape, a 5′ end, which, being unstructured, may be a very efficient but it uses a very different set of intramolecular interactions leader to allow detectable translation of CP (or Rap1) ORF (Colussi et al., 2015). These interactions allow the TLS to switch from undetectable amounts of degraded RNA. This alternative conformations and to interact with the ribosome, docking within mechanism of expression may still be biologically relevant, or it to regulate the folding and unfolding state to permit dual simply an artifact of the assays, but would not result from an functionality in viral translation and replication. This leads us to IRES. hypothesize that TLS recruits the ribosome, which is delivered ′ ′ ′ to the 5 -UTR by communication with the 5 end through the Enhancers Located in the 3 -UTR cap-eIF4E-eIF4G-eIF3-40S chain of interactions. tRNA-Like Structures A different function for tRNA mimicry occurs in the only Viruses from the family Bromoviridae and the genera IRES that occurs naturally between ORFs: the intergenic region Tobamovirus and Tymovirus possess a 5′ cap structure but (IGR) IRES of dicistroviruses (Wilson et al., 2000; Khong et al., lack a 3′ poly(A) tail. In contrast, they contain tRNA-like 2016). In the IGR IRES, a pseudoknot mimics the structure of structures (TLSs) at their 3′ termini that perform many viral the anticodon loop of a tRNA basepaired to a codon in mRNA, processes, such as (i) serving as a telomere by interacting facilitating instant elongation as the ribosome joins the viral RNA with CTP:ATP nucleotidyl transferase which adds CCA in a with no initiation steps (Costantino et al., 2008). non-templated fashion to the 3′ end (Rao et al., 1989), (ii) In the case of BMV RNA, its 3′-UTR has been shown to regulation of negative strand synthesis (Dreher, 2009), (iii) provide translation enhancement, and the disruption of its TLS translation enhancement (Gallie and Walbot, 1990; Choi et al., reduced translation in vitro (Barends et al., 2004). On the other 2002; Matsuda and Dreher, 2004), and (iv) packaging of the hand, the TMV TLS is structurally similar to the TYMV TLS and viral RNA in the virion (Annamalai and Rao, 2007). Three basic functions as minus-strand promoter (Chapman and Kao, 1999), types of 3′ terminal TLS have been described in the genomes of but it does not mediate translation enhancement. However, the Turnip yellow mosaic virus (TYMV; genus Tymovirus, family 3′-UTR of TMV contains an upstream pseudoknot domain that Tymoviridae), TMV and Brome mosaic virus (BMV; genus stimulates translation in a way that is replaceable by a poly(A) Bromovirus, family Bromoviridae). Because of their multiple tail (Gallie et al., 1991; Leathers et al., 1993). Additionally, TMV functions, it has been difficult to tease out the mechanisms of RNA also harbors in its 5′-UTR the 68-nt omega () sequence each role, but the translational enhancement structures and which highly stimulates cap-dependent translation (Gallie and mechanisms have been well characterized for TMV and TYMV Kado, 1989).  is recognized by the heat shock protein 101 (Table 1). (HSP101), mediating translational activity (Wells et al., 1998) and The TYMV TLS requires aminoacylation of the 3′-CCA interacts with eIF4F via eIF4G (Gallie, 2002, 2016). Similarly, terminus for maximal translational efficiency and the 5′ cap the Brassicaceae-specific eIFiso4G2 isoform also contributes in synergistically promotes this activity (Matsuda et al., 2004). -mediated translation, unlike eIFiso4G which did not affect - Translational enhancement maps principally to the TLS, dependent translation (Gallie, 2016). These results suggest that although the upstream adjacent pseudoknot is important for eIFiso4G2 exhibits more functional similarity with eIF4G than optimal translation, possibly serving as a sequence spacer eIFiso4G. Regarding translational activity,  is one of the most (Matsuda and Dreher, 2004). The aminoacylated TLS binds to efficient mRNA leaders in vitro and in vivo and it was used eukaryotic elongation factor 1A (eEF1A) and is a substrate for for biotechnological applications such as transgene expression tRNA-modifying enzymes (Dreher and Goodwin, 1998; Matsuda (Gallie et al., 1987; Fan et al., 2012). et al., 2004) mimicking tRNA activity. The 5′-proximal AUG ′ in the TYMV genome serves as start codon for a 69 kDa ORF 3 -UTR Mediated Translation of the Alfalfa mosaic (p69), and the second AUG is the start codon for the main virus Genome polyprotein ORF (p206) with which ORF p69 overlaps. Based on The non-polyadenylated Alfalfa mosaic virus (AMV, genus only in vitro translation assays, Barends et al. (2004) proposed Alfamovirus, family Bromoviridae) RNA requires the viral CP for a “Trojan Horse” model of translation initiation in which the efficient translation and infection. The 3′-UTR of AMV also plays aminoacylated TLS delivers its amino acid to the start codon a role in translation due to its ability to bind the CP, adopting the of the polyprotein ORF. However, the Dreher lab provided in CP-binding (CPB) conformation. This binding avoids the minus- vitro and in vivo evidence that a more likely mechanism is strand promoter activity and enhances translation, possibly classical leaky scanning, except that the efficiency of initiation acting as a mimic of the poly(A) tail (Olsthoorn et al., 1999). The at the second AUG correlated with its proximity to the first CPB structure folds into a series of stem-loops separated by an AUG (Matsuda and Dreher, 2006). In addition, the translation AUGC motif and mutations in this motif led to the loss of binding efficiency of the polyprotein ORF depended on a 5′ cap, and to CP, correlating with reduction of translation in protoplasts not the 3′ TLS. This and additional data support an “initiation (Reusken and Bol, 1996; Neeleman et al., 2004). The crystal coupling” model in which the close proximity (7 nt) of the structure of CP-bound RNA revealed a novel RNA fold in which two AUG codons is necessary for maximum translation of the RNA forms two hairpins separated by the linker AUGC motif and polyprotein ORF (Matsuda and Dreher, 2007). oriented in right angles (Guogas et al., 2004). The presence of the How the TLS interacts with the 5′ end to stimulate translation CP promotes the base pairing between linker motifs, leading a in the scanning-dependent manner is suggested by the crystal compact structure. Moreover, pulldown assays revealed that the

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CP interacts with eIF4G/eIFiso4G subunits (Krab et al., 2005). p27 ORF for efficient translational activity (Blanco-Pérez et al., This interaction may stimulate mRNA circularization in a similar 2016). fashion as found for rotaviruses (Groft and Burley, 2002). In The shortest CITEs are the I-shaped structures (ISS) present in addition to the CPB form, AMV RNAs 3′ termini also fold into the 3′-UTRs of Maize necrotic spot virus (MNeSV, Tombusvirus, a pseudoknot structure that resembles a TLS conformation. The family Tombusviridae) and Melon necrotic spot virus (MNSV, 3′-UTR can be recognized by a tRNA-specific enzyme and by the genus Carmovirus, family Tombusviridae)(Truniger et al., 2008; viral replicase and this recognition is inhibited by the addition of Nicholson et al., 2010; Miras et al., 2016), and are apparently CP (Olsthoorn et al., 1999; Chen and Olsthoorn, 2010). Thus, it similar in secondary structure to the TED. MNeSV ISS has been suggests that TLS conformation acts as a minus-strand promoter shown to preferentially interact with the eIF4E subunit of eIF4F. and the CP interaction and pseudoknot stability may regulate a As for TED and most other CITEs, base pairing between the 3′- conformational switch between translation and replication (Chen CITE and the 5′-UTR is predicted to deliver the translation factor and Olsthoorn, 2010). to the 5′ end, facilitating recruitment of the 43S preinitiation complex (Nicholson et al., 2010). In support of this model, it Cap-Independent Translation Elements has been shown that the interacting 5′-UTR:I-shaped 3′-CITE of Members of the Tombusviridae and Luteoviridae plant virus MNeSV together with eIF4F form a complex in vitro. In addition, families lack both 5′ cap and 3′ poly(A) elements, but contain ribosome toe printing demonstrated that while bound to eIF4F, in their 3′ ends structured RNA elements capable of enhancing the I-shaped CITE can simultaneously base pair with the 5′- translation in the absence of cap (cap-independent translation UTR and recruit ribosomes to the 5′ end of the viral fragment elements, CITEs). Most 3′-CITEs have in common their ability to (Nicholson et al., 2010). bind translation initiation factors of the eIF4E or eIF4G families, In the case of MNSV ISS, genetic evidence for interaction of as well as the presence of small sequence stretches within or near the ISS with eIF4E has been shown in melon. A single amino acid the 3′-CITE capable of base-pairing to sequences in the 5′-UTR change in melon eIF4E strongly reduces translation efficiency of the mRNA to establish long-distance RNA:RNA interactions controlled by MNSV ISS and makes melon resistant to MNSV (Table 1). By definition, 3′-CITEs functionally substitute for the infection (Nieto et al., 2006; Truniger et al., 2008). The minimal 5′ cap with high efficiency. They recruit translation initiation 3′-CITE sequence, named Ma5TE (MNSValpha5-like translation factors leading to ribosome entry at or near the 5′ terminus enhancer), was mapped to a 45 nt region. In vitro binding followed by ribosome scanning to the initiation codon (Fabian assays revealed that Ma5TE forms a complex with eIF4F and this and White, 2004; Rakotondrafara and Miller, 2008; Nicholson interaction was mapped to a conserved guanosine residue located and White, 2011); therefore, in contrast to IRESes, 3′-CITEs in a Ma5TE internal loop (Miras et al., 2016). Additionally, do not promote internal ribosome entry. To date, seven mutational analyses in eIF4E residues involved in its interaction different classes of 3′-CITEs have been described (Simon and with eIF4G showed that eIF4F complex formation is necessary for Miller, 2013; Miras et al., 2014) which share little secondary efficient cap-independent translation driven by Ma5TE (Miras structure and sequence similarity. Due to space limitations and a et al., 2016). Identification of a new resistant-breaking isolate previous comprehensive review on 3′-CITEs (Simon and Miller, of MNSV revealed a new class of 3′-CITE, the CXTE, which 2013), we will describe only briefly each 3′-CITE and recent was acquired from Cucurbit aphid-borne yellows virus (CABYV, updates. genus Polerovirus, family Luteoviridae) Xinjiang by interfamilial The first 3′-CITE was discovered in Satellite tobacco necrosis recombination, conferring to the recipient MNSV isolate the virus (STNV) and is located in a 120-nt sequence termed advantage to translate efficiently and infect resistant melon translation enhancer domain (TED) (Danthinne et al., 1993; varieties (Miras et al., 2014). Thus, the 3′-UTR of this MNSV Timmer et al., 1993; Meulewaeter et al., 1998). The TED is isolate harbors two 3′-CITEs, Ma5TE, and CXTE, with CXTE predicted to form a long stem-loop with several internal bulges secondary RNA structure folding into two helices protruding (Van Lipzig et al., 2002). This element was shown to be functional from a central hub. Both 3′-CITEs are active in susceptible melon, in enhancing translation in vitro and in vivo. TED binds eIF4F or while only the CXTE functions in resistant melon and in the eIFiso4F (Gazo et al., 2004), and is proposed to interact with the absence of eIF4E (Miras et al., 2014). 5′-UTR via a predicted RNA:RNA long-distance interaction with The Barley yellow dwarf virus-like translation element (BTE) the apical loop of the 5′ end. However, mutations that disrupted is one of the best-characterized 3′-CITEs and is found in this potential long-distance base-pairing reduced translation only all members of the Luteovirus, Dianthovirus, Alphanecrovirus, slightly, and covarying mutations designed to restore base pairing Betanecrovirus, and Umbravirus genera (Wang et al., 2010; did not restore translation to wild type levels (Meulewaeter et al., Simon and Miller, 2013). All BTEs share a long basal helix 1998). The STNV 3′-CITE confers cap-independent translation from which two to five additional helices radiate (Figure 1). in vitro when it is moved to the 5′-UTR of an uncapped BTEs contain a highly conserved 17-nucleotide sequence reporter (Meulewaeter et al., 1998). Another member of the GGAUCCUGGGAAACAGG that includes SL-I (formed by Tombusviridae family, Pelargonium line pattern virus (PLPV, pairing of underline bases). The BTE binds preferentially to genus Carmovirus) was recently shown to harbor a 3′-CITE in the eIF4G subunit of the eIF4F heterodimer (Treder et al., the TED class (Blanco-Pérez et al., 2016). In this case, PLPV TED 2008). The eIF4G-binding site in the BTE was revealed by was shown to require a long-range RNA:RNA kissing stem-loop SHAPE footprinting, which showed that eIF4G protects SL- interaction with a hairpin in the coding sequence of the PLPV I and nearby bases around base of the hub from which all

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FIGURE 1 | Non-canonical initiation translation mechanisms used by plant RNA viruses. Canonical translation of eukaryotic mRNAs is shown in the top. Non-canonical translation elements are grouped depending on their location in viral genome and are color-coded to match with the virus acronyms. Lighter-shaded loops in the secondary structure of 3′-CITEs indicated sequences known or predicted to base-pair to the 5′ end of the viral genome (shown as dashed line). helices protrude (Kraft et al., 2013). Addition of eIF4E enhanced After eIF4F binds the BTE, it appears that the eIF4A helicase, the level of protection and stimulated translation by about eIF4B plus ATP bind in order to recruit the 40S subunit directly 25%. Deletion analysis of eIF4G revealed that only the core to the BTE. The long-distance base pairing would then deliver the domain (including eIF4A and eIF3 binding sites, but lacking 40S complex to the 5′ end for scanning to the first AUG (Sharma the eIF4E and PABP binding sites) and an adjacent upstream et al., 2015; Figure 2A). This differs from a previous model in RNA binding domain are necessary for binding to the BTE which it was proposed that the long-distance base pairing places and to stimulate translation (Kraft et al., 2013; Zhao et al., the factors near the 5′ end, at which point the 40S complex is 2017). recruited (Rakotondrafara et al., 2006). However, the dependence A long-distance kissing stem-loop interaction between a on helicase activity may support an older model in which a six loop in the BTE and the 5′-UTR is required for BTE- base tract in the 17 nt conserved sequence (GAUCCU) base pairs mediated translation (Guo et al., 2001). This long-distance directly to 18S rRNA at the position where the Shine-Dalgarno RNA:RNA interaction can be replaced by complementary sequence is located in prokaryotic ribosomal RNA (Wang et al., non-viral sequences outside the BTE (Rakotondrafara et al., 1997). Because much of this tract is base paired internally in both 2006). This interaction is conserved among all BTEs except the BTE and in 18S rRNA, the helicase activity may be required to the BTE of Red clover necrotic mosaic virus (RCNMV, genus disrupt this base pairing, freeing the complementary tracts in the Dianthovirus, family Tombusviridae), in which mutations in BTE and 18S rRNA to base pair to each other. This base pairing potential complementary loops had no effect on translation and would recruit the 40S subunit directly to the BTE (Figure 2A). possess the longest BTE and 3′-CITE (Sarawaneeyaruk et al., However, recently the presence of eIF4A, eIF4B and ATP was 2009). also found to enhance the binding affinity of the BTE to eIF4G

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′ FIGURE 2 | Alternative models of ribosome recruitment and delivery to the 5 -UTR via the BTE. (A) Base pairing to rRNA model. Top: eIF4F binds to SL-I of the BTE (green) through the eIF4G subunit. eIF4E enhances but is not required for BTE binding. Middle: Helicase (eIF4A + eIF4B) binds and uses ATP hydrolysis to unwind GAUCCU, making it available to base pair to 18S rRNA at a conserved sequence in the region where the Shine-Dalgarno binding site is located in prokaryotic 16S rRNA. Bottom: The 43S preinitiation complex base pairs to the BTE and is delivered to the 5′ end by long-distance base pairing (yellow stem-loop). (B) Conventional ribosome recruitment model. Top: eIF4F binds BTE as in (A). Middle: Binding of eIF4A + eIF4B and ATP hydrolysis increases binding affinity of eIF4F, “locking” it on to the BTE, perhaps by altering the structure of BTE RNA. Bottom: eIF4 complex is delivered to 5′ end by long-distance base pairing where it recruits the 43S preinitiation complex to the RNA. In both models, 43S scanning from the 5′ end to the start codon is the same as in normal cap-dependent translation. Not shown: other factors, such as eIF3 and factors in the preinitiation complex. in the absence of the ribosome (Zhao et al., 2017). This enhanced (YSS), formed by three helical regions. The efficiency of binding affinity may be the consequence of helicase activity of translation controlled by the YSS of TBSV depends on a long- eIF4A/eIF4G/ATP altering BTE structure. This greater affinity distance interaction with the 5′-UTR of the genome. Mutational of eIF4G to the BTE may facilitate efficient recruitment of the analysis of TBSV YSS showed that alterations in junction 40S subunit by conventional factor interactions without need for residues between helices and in a large asymmetric bulge in the base pairing to ribosomal RNA (Figure 2B). Future experiments major supporting stem disrupted translation (Fabian and White, are necessary to determine which model is correct. On the other 2004, 2006). Moreover, the YSS of Carnation Italian ringspot hand, RCNMV possesses an A-rich sequence (ARS) with strong virus (CIRV, genus Dianthovirus, family Tombusviridae) requires affinity to PABP in addition to its BTE in its 3′-UTR. Both addition of the eIF4F or eIFiso4F complex to a factor-depleted sequences, ARS and 3′-CITE, have been shown to coordinately wheat germ extract to promote efficient translation (Nicholson recruit eIF4F/ eIFiso4F and the 40S ribosomal subunit to the viral et al., 2013). Translation assays showed the ability of the CIRV RNA (Iwakawa et al., 2012). YSS to function efficiently in vitro and in vivo, whereas TBSV Tomato bushy stunt virus (TBSV, genus Tombusvirus, family YSS was detectable only in in vivo, suggesting that this difference Tombusviridae) and other viruses belonging to the genus is due to a misfolding in the TBSV RNA and the lack of eIFs Tombusvirus, contain 3′-CITEs resembling Y-shaped structure required in translation (Fabian and White, 2004).

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′ ′ The Panicum mosaic virus-like Translation Enhancer (PTE) 5 - and 3 -UTR Dependent Translation of was first identified in Panicum mosaic virus (PMV, genus Nepovirus Genomic RNAs Panicovirus, family Tombusviridae)(Batten et al., 2006) and later As mentioned above, nepovirus (family Secoviridae, order in Pea enation mosaic virus 2 (PEMV2, genus Umbravirus, family Picornavirales)genomescontainaVPglinkedtotheir5′ end, thus Tombusviridae)(Wang et al., 2009b). The PEMV2 PTE consists are uncapped but polyadenylated requiring also cap-independent of a three-way branched helix with a large G-rich bulge in the translation mechanisms. The two genomic RNAs (gRNA) of main stem (Wang et al., 2009b). The formation of a magnesium- Blackcurrant reversion virus (BRV; genus Nepovirus, subfamily dependent pseudoknot between the G-rich bulge and a C-rich Comoviridae), have translation enhancing sequences in their sequence at the three-helix junction of the PTE is critical for 5′- and 3′-UTRs. The 5′ leader sequences of the two gRNAs translation and eIF4E recruitment by the PTE (Wang et al., of BRV contain IRES elements that facilitate translation when 2011). Unlike most other CITEs, the PEMV2 PTE may not ′ ′ placed either at the 5 -end of a non-capped reporter RNA or participate in a long-distance RNA:RNA interaction with the 5 - internally between two reporter genes (Karetnikov and Lehto, UTR. Instead, upstream of the PTE, there is an element, the 2007, 2008). The BRV IRESes contain little secondary structure, kl-TSS, that participates in a long range RNA:RNA interaction ′ ′ harboring only one predicted single stem-loop structure at the5 with a 5 proximal hairpin located in the p33 ORF (Gao et al., end. Also, the 5′-UTRs of both gRNAs have at least six AU-rich 2012). tracts of 8–10 nt predicted to base pair to 18S rRNA. Deletion Most other PTEs contain a loop predicted to base pair to the ′ of these sequences reduced cap-independent translation activity, 5 -UTR. Indeed, Saguaro cactus virus (SCV, genus Carmovirus, suggesting a disruption of the required complementarity or other family Tombusviridae), harbors a PTE which participates in a 5′-UTR functional features (Karetnikov and Lehto, 2007, 2008). long-distance RNA:RNA interaction with a hairpin located in the In addition to these IRESes, CITE activity was mapped to p26 ORF (Chattopadhyay et al., 2011). Interestingly, the sequence the 3′-UTRs of BRV RNA1 and RNA2 (Karetnikov et al., 2006; involved in the interaction has the same conserved motif found Karetnikov and Lehto, 2008). This activity depended on the in carmovirus TED-like elements and I-shaped structures (Simon presence of a predicted stem-loop structure located immediately and Miller, 2013). ′ downstream of the last ORF. Moreover, translation efficiency The 3 -UTR of another member of the Tombusviridae was shown to be dependent on a long-distance RNA interaction family, Turnip crinkle virus (TCV, genus Carmovirus, family with a stem-loop structure present in the 5′-UTR (Karetnikov Tombusviridae), contains an internal T-shaped structure (TSS) and Lehto, 2008). Secondary structures of the 3′-CITE and 5′- that consists of three hairpins, two pseudoknots and multiple UTR have not been determined, although they are predicted to unpaired single stranded linker regions (Zuo et al., 2010). fold as a pseudoknot and a stem-loop, respectively. Presence of a Interestingly, the TSS resembles a three-dimensional tRNA-like poly(A) tail (which is naturally present in the BRV RNA, unlike in structure (Zuo et al., 2010). The TCV TSS recruits and binds other 3′-CITE-containing viruses) in reporter mRNAs stimulated the 60S subunit of the 80S ribosome (Stupina et al., 2008). For ′ ′ translation several fold, thus playing a major role in CITE- this element, no base pairing between 3 -CITE and 5 -UTR has mediated translation. Many of the key elements identified in been identified. It was proposed that the ribosomal subunits form BRV RNAs, including 5′–3′-UTR RNA interactions and sequence a protein bridge with the UTRs, where the 40S subunit binds ′ ′ complementarity with the 18S rRNA in the 5 -UTR, are predicted the 5 -UTR and the 60S subunit binds the TSS (Stupina et al., to be conserved in the RNAs of other nepoviruses (Karetnikov 2008). Two additional TSSs were found in the PEMV2 3′-UTR, ′ and Lehto, 2008), although their precise biological functions one upstream of the PTE and another near to the 3 terminus remain unknown. (Gao et al., 2013). Interestingly, both TSSs can also bind the 60S ribosomal subunit and although they are essential for virus accumulation in vivo, mutations that disrupted the downstream OPTIMIZATION OF CODING CAPACITY TSS had no effect in translation (Gao et al., 2013, 2014). However, when this TSS element was positioned proximal to the reporter RNA viruses often contain overlapping genes, which allows a very ORF enhanced translational activity. This report points out the efficient use of the sequence to maximize the coding capacity. importance of the reporter constructs in the identification of Expression of these overlapping genes is achieved by (i) initiation 3′-CITE that participate in translation. A recent report showed of translation at multiple start codons in different reading frames, that TCV RdRp binds to A-rich sequence upstream of the by leaky scanning of ribosomes, (ii) frameshifting by a portion TSS and using optical tweezers and steered molecular dynamic of the ribosomes during the elongation phase of translation, or simulations showed that elements of TSS unfold when it is (iii) generating subgenomic mRNAs that allow translation of each interacting with RdRp which may promote the conformational ORF from a separate mRNA. The latter will not be discussed, as switch between translation and replication (Le et al., 2017). it is not a translational control mechanism. More classes of 3′-CITE await discovery, as the 3′ UTRs of several members of the Tombusviridae contain no structure that Leaky Scanning obviously resembles a known 3′-CITE (Simon and Miller, 2013). Leaky scanning occurs when a proportion of ribosomes fail Thus, viruses have evolved a plethora of structures to achieve the to initiate translation at the first AUG codon and continue same goal: recruitment of eIF4F and ultimately the ribosome to downstream until they reach an AUG codon in the optimal their RNAs. caA(A/C)aAUGGCg initiation context (Figure 3; Joshi et al.,

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be facilitated by the use of non-AUG initiation codons, which require a strong initiation context (Kozak, 1989). In this respect, Shallot virus X (ShVX; family Flexiviridae, genus Allexivirus) contains a non-canonical ORF for its TGB3 protein (Kanyuka et al., 1992; Lezzhov et al., 2015). ShVX TGB3 translation initiates in a CUG triplet, which has been shown previously to be the most efficient non-AUG initiator (Firth and Brierley, 2012). This triplet and flanking sequences give an optimal context for translation initiation and are conserved in all allexviruses (Lezzhov et al., 2015). Similarly, translation of the second movement protein from Pelargonium line pattern virus (PLPV, family Tombusviridae, genus Pelarspovirus) and Maize chlorotic mottle virus (MCMV, family Tombusviridae, genus Machlomovirus) were suggested to be initiated in a GUG or CUG start codons, accomplished by leaky scanning (Scheets, 2000; Hernández, 2009). In the main subgenomic RNA of poleroviruses and luteoviruses all three reading frames are used. The tiny, 45 codon first ORF, which encodes a long-distance movement protein, always starts with a non-AUG codon, such as GUG, CUG or AUU. Thus, most scanning 40S ribosomes skip this codon (Smirnova et al., 2015). The second ORF, which encodes the coat protein, starts with AUG in a poor context, while the third ORF, a movement protein gene, starts with AUG in a strong FIGURE 3 | Viral recoding strategies. Top panel represents leaky scanning context. The secondary structure encompassing these two AUGs mechanism where ribosomes fail to start translation at the first AUG codon also affects initiation preference (Dinesh-Kumar and Miller, and continue scanning until they reach an alternative start codon in the optimal 1993). Other examples of leaky scanning in replicase ORFs initiation context. This process allows the expression of two proteins with distinct amino acid sequence when the initiation sites are in different reading have been described. In tymoviruses, the first AUG initiates an frames (as shown) or C-terminally coincident isoforms of a single protein if ORF encoding a 69 kDa protein that overlaps with the main initiation sites are in-frame (not shown). Middle panel shows the expression of replicase-encoding ORF initiated by the second AUG. While proteins with alternative C-terminal because a portion of ribosomes fail to Kozak context plays a role, unlike “conventional” leaky scanning, terminate at a stop codon and continue translation. The efficiency of readthrough can be stimulated by the presence of elements downstream of the second AUG must be in close proximity (e.g., 7 nt) of the the stop codon: UAG stop codon followed by the consensus motif CARYYA, first to efficiently initiate translation (Matsuda and Dreher, where R is a purine and Y is a pyrimidine (Type I); UGA stop codon followed by 2006). Recently, a small ORF in the sobemoviruses, ORFx, was CGG or CUA triplet and a stem-loop structure separated from the stop codon discovered that overlaps ORF2a and is essential for Turnip rosette by 8 nt (Type II); UAG stop codon and adjacent G or purine octanucleotide and virus (TRoV, genus Sobemovirus) to establish systemic infection a compact pseudoknot structure (Type III). Bottom panel represents ribosomal frameshifting strategy, where ribosomes are directed into a different reading (Ling et al., 2013). frame guided by the slippery signal X_XXY_YYZ (X and Y can be any base and Z is any base except G) and a secondary structure element located 5-9 nt Translational Recoding: Frameshift and downstream the slippery sequence. Readthrough Recoding consists of the redefinition of individual codons in response to signals in an mRNA. Such signals could be RNA secondary structures, complementary interactions with 1997; Kozak, 2002). If the two AUG codons are in the same ribosomal RNA or alteration of the ribosomal state (Atkins reading frame, the protein derived by initiation at the second and Baranov, 2010). In ribosomal frameshifting a proportion of AUG is an N-terminally truncated version of that made by translating ribosomes are guided into a different reading frame initiation at the first AUG. If the two AUGs are in different by induced slippage of the ribosome by the mRNA structure frames, then the two proteins have entirely different amino (exhaustively reviewed by Miller and Giedroc, 2010; Atkins et al., acid sequences. Examples of the latter are long overlaps of 2016), while in readthrough mechanisms, a portion of ribosomes replication genes and the triple gene block (TGB) that encodes fail to terminate at a stop codon and continue translation the movement proteins of several viruses: For instance, the TGB3 (Figure 3). This generates proteins with alternative C-termini. of Potato virus X (PVX, family Flexiviridae, genus Potexvirus) Viruses use often these processes to express the RNA-dependent and Barley stripe mosaic virus (BSMV, family Virgaviridae, RNA polymerase domain of the replicase. genus Hordeivirus) and the TGB2 of Peanut clump virus (PCV, family Virgaviridae, genus Pecluvirus) are expressed by Ribosomal Frameshifting leaky scanning (Herzog et al., 1995; Zhou and Jackson, 1996; Many plant viruses utilize programmed ribosomal frameshifting Verchot et al., 1998). In addition, leaky scanning may also (PRS) to translate overlapping ORFs. This recoding event can

Frontiers in Plant Science | www.frontiersin.org 11 April 2017 | Volume 8 | Article 494 Miras et al. Non-canonical Translation in Plant RNA Viruses occur in the + or − direction relative to the normal 0 frame Stop-Codon Readthrough of mRNA translation by shifting the ribosome in one or Stop-codon readthrough is a common strategy found in plant two nucleotides forward or backward. Productive frameshifting viruses to encode protein variants with an extended C-terminus normally competes poorly with standard decoding, so the from the same RNA. During readthrough, some ribosomes efficiency of frameshifting in viruses varies from 1% in BYDV to do not stop at the stop codon but continue until the next 82% in cardioviruses (Barry and Miller, 2002; Finch et al., 2015). termination codon. Members of the Tombusviridae, Luteoviridae Thus far, most frameshifting by plant viruses is in the -1 direction. and Virgaviridae families employ readthrough of UGA and UAG These include members of the Sobemovirus, Umbravirus, and stop codons in their replicase and coat protein genes. Flanking Dianthovirus genera and the Luteoviridae family (Brault and nucleotides as well as long-range RNA-RNA interactions Miller, 1992; Demler et al., 1993; Kujawa et al., 1993; Mäkinen influence stop-codon readthrough (Figure 3; Firth and Brierley, et al., 1995; Kim et al., 1999; Lucchesi et al., 2000; Barry 2012; Nicholson and White, 2014). Depending on the sequence and Miller, 2002; Tamm et al., 2009). Members of the non- motifs and the stop codon, three types of readthrough can be related family Closteroviridae (genus Closterovirus, Crinivirus described: The type I motif employs a UAG codon in the replicase and Ampelovirus) are predicted to use a +1 frameshift to gene and is followed by the consensus motif CARYYA (where synthesize their viral replicases (Agranovsky et al., 1994; Karasev R is a purine and Y is a pyrimidine) (Skuzeski et al., 1991); et al., 1995; Melzer et al., 2008). this type is used by tobamoviruses, benyviruses and pomoviruses The -1 PRS usually requires two signals in the mRNA, a (Pelham, 1978; Firth and Brierley, 2012). The type II motif is slippery sequence of the type X_XXY_YYZ, where X normally used by tobraviruses, pecluviruses, furoviruses and pomoviruses represents any nucleotide, Y represents A or U and Z represents to generate their viral RdRp and by furoviruses to express the coat A, C or U (gaps delimit codons in the original 0 frame); and protein (Skuzeski et al., 1991; Zerfass and Beier, 1992). It involves a downstream secondary structure element separated from the a UGA stop codon followed by a CGG or CUA triplet and a stem- slippery sequence by a spacer region of 5-9 nt (Dinman, 2012). loop structure about 8 nts downstream of the stop codon (Firth In plant viruses these structural elements, acting as stimulators et al., 2011). The type III class comprises an UAG stop codon, of frameshifting, fall into three structural classes: an apical loop a downstream G or purine-rich octanucleotide and a 3′ RNA with a bulge, a compact hairpin-type pseudoknot or a stem-loop structure (Firth and Brierley, 2012) and appears in carmovirus (Figure 3) (reviewed by Miller and Giedroc, 2010). and tombusvirus genomes. For example, the tombusvirus CIRV The -1 PRS stimulatory elements of BYDV, PEMV-RNA2 uses stop-codon readthrough to generate its viral RdRp and and RCNMV fold into a stem-loop with an internal bulge in a requires a long-distance interaction between an RNA structure similar manner (Kim et al., 1999; Paul et al., 2001; Barry and located downstream of the readthrough site and also a sequence Miller, 2002; Gao and Simon, 2015). For BYDV, this element in the 3′-UTR (Cimino et al., 2011). Tobacco necrosis virus-D participates in a long-distance interaction with the apical loop (TNV-D, genus Betanecrovirus, family Tombusviridae) employs of a stem-loop located in the 3′-UTR (about 4 kb downstream a complex series of downstream interactions. A stable bulged of the frameshift site). This interaction is required for the low readthrough stem-loop (RTSL) immediately downstream of the expression levels of RdRp and thus replication (Barry and Miller, leaky stop codon contains a G-rich bulge which must base 2002). Similar long-range base pairing interactions were shown pair to a distant readthrough element (DRTE) located 3 kb in RNAs of RCNMV and PEMV2 (Tajima et al., 2011; Gao and downstream in the structure required for replication initiation Simon, 2015). For PEMV2 RNA, this interaction modifies the (Newburn et al., 2014). A pseudoknot immediately 3′ to the lower stem of the structure, possibly due to a rise of its stability or RTSL, and a stem-loop adjacent 5′ to the DRTE in the 3′- the approximation of other sequence near the 3′ end. Curiously, UTR are also necessary for optimal readthrough (Newburn and the distant −1 PRS element of PEMV2 RNA appeared to inhibit, White, 2017). The long-distance interactions within the viral rather than stimulate frameshifting, because in its absence, the genome required for frameshifting and readthrough may play frameshift rate increased 72% with respect to the wild type viral a regulatory role as switch between translation and replication genome (Gao and Simon, 2015). (Cimino et al., 2011), by allowing replicase entering at the 3′ On the other hand, the frameshift stimulatory elements from end of the genome to stop its own translation 3–4 kb upstream, poleroviruses Beet western yellows virus, Potato leaf roll virus as it disrupts this essential long-distance interaction (Miller and and Sugarcane yellow leaf virus (BWYV, PLRV, and ScYLV, White, 2006). family Luteoviridae, genus Polerovirus) and PEMV1 (family Readthrough of the CP stop codon of viruses from the Luteoviridae, genus Enamovirus) form h-type pseudoknots (Egli Luteoviridae family appears to use a fourth class of cis-acting et al., 2002; Cornish et al., 2005; Pallan et al., 2005; Giedroc signals (Brown et al., 1996). The stop codon is usually UAG, and Cornish, 2009). The frameshift regulatory element of BWYV but can be UGA or UAA. Instead, readthrough requires a tract was the first to be determined at atomic resolution showing a of 8–16 repeats of CCXXXX beginning about 8 nt downstream compact pseudoknot with a triple-stranded region (Egli et al., of stop codon and requires additional sequence about 700–750 2002). It was suggested that pseudoknots provide a kinetic nt downstream in the coding region of the readthrough ORF, barrier to the ribosome and that the unfolding of this element in the example of BYDV (Brown et al., 1996). Although the correlates with frameshifting stimulation (Giedroc and Cornish, resulting CP-readthrough protein fusion is not essential for virus 2009). particle assembly or infectivity it is assembled into the virion and

Frontiers in Plant Science | www.frontiersin.org 12 April 2017 | Volume 8 | Article 494 Miras et al. Non-canonical Translation in Plant RNA Viruses is required for persistent, circulative aphid transmission (Brault of experiments covering biochemistry, genetics and cellular et al., 1995; Chay et al., 1996). biology are also missing. For instance, wheat germ extract has been and still is very useful for biochemistry experiments, PERSPECTIVES but genetics or cellular biology experiments are difficult using wheat as a host, because it is hexaploid and difficult to This review provides an outlook of the vast diversity of transform. In another example, N. benthamiana is an excellent non-canonical mechanisms that RNA viruses use to translate host to perform cellular biology experiments, but its genetic their RNAs. With some significant exceptions, knowledge is tractability is rather poor, and N. benthamiana is not particularly still superficial for a large number of cases. It would be advantageous for biochemistry experiments. In this regard, the highly desirable to obtain additional and deeper information preparation of translationally active extracts from evacuolated on specific cases and mechanisms. For example, secondary protoplasts (Murota et al., 2011) from different plant species structure data is available for only a few translation initiation may contribute to solve this problem, particularly if prepared elements (Wang et al., 2009a; Zuo et al., 2010; Nicholson from genetically tractable and microscopy amenable hosts such and White, 2011; Kraft et al., 2013; Miras et al., 2014), and as Arabidopsis. high-resolution three-dimensional structures are known only From the point of view of the cellular translational machinery for the small H-type pseudoknot frameshift structures of the and how viruses use it, the described diversity of translation polero- and enamoviruses (Miller and Giedroc, 2010), and for mechanisms points toward the different ways that viruses use plant translation factor eIF4E (Monzingo et al., 2007; Ashby and control the basic translation machinery of the cell, but et al., 2011). There is no structural data on bipartite or it also seems to point toward the existence of a diversity of multipartite virus-host complexes. This represents a significant associations of RNA and protein translation factors used for methodological challenge, but the current advancement of the uninfected cell to synthesize proteins from different mRNA techniques like cryo-electron microscopy may significantly populations under different micro-environmental conditions contribute to tackle it. Structural data would provide additional and/or subcellular locations. It is tempting to speculate that mechanistic insight and could contribute to uncover interacting during evolution plant viruses may have adopted cellular regions with regulatory roles, providing molecular targets for preexisting mechanisms to translate their proteins; quite likely, intercepting productive host-virus interactions. there is a significant overlap between translation mechanisms It is important to note that mutations in translation initiation of viral RNAs and translation of cellular mRNAs in uninfected factors that disrupt interactions with viral proteins or RNA might cells under abiotic stress conditions (Spriggs et al., 2010), and not only prevent infection in resistant varieties of susceptible host viruses might be viewed as useful probes to uncover the cellular species but also contribute to non-host resistance. Mutations in mechanisms of translation. In this regard, it has been shown viral factors conferring compatibility with translation initiation that active plant virus replication associates with host gene factors of otherwise non-host plants can contribute to broaden shutoff (Wang and Maule, 1995; Aranda and Maule, 1998), but the host range of potyviruses (Calvo et al., 2014; Estevan et al., even during active replication there are host mRNAs which are 2014; Svanella-Dumas et al., 2014) and a carmovirus (Nieto over-expressed, suggesting common mechanisms for host and et al., 2011). Also, the development of techniques to monitor viral mRNA expression, including translation; structural data the translational dynamics based on fluorescent and optical may provide important information on how these transcripts methods could provide more complete pictures of how and where can recruit the host machinery efficiently during cellular shut- translation occurs. off. Interestingly, host mRNAs over-expressed during virus The diversity of mechanisms is particularly striking when replication include stress response transcripts (Aranda et al., identified in a single viral RNA. BRV provides an example 1996) and, in fact, at least a maize HSP101 and ADH1 of this, with gRNAs carrying VPg, poly(A), IRES, and CITE transcripts have been shown to contain IRES-like elements (Karetnikov et al., 2006; Karetnikov and Lehto, 2008), and (Dinkova et al., 2005; Mardanova et al., 2008). Large screenings there are other viral RNAs for which multiplicity of cis-acting of the human genome revealed widespread identification of cap- elements has been recognized, including MNSV (Miras et al., independent translation elements located in the 5′-UTR and 2014) and PEMV2 (Gao et al., 2013, 2014). This multiplicity 3′-UTR of human transcripts, but their mode of regulation may exist for different reasons, including the use of different remains unknown (Weingarten-Gabbay et al., 2016). In plants, mechanisms during different steps of the infection cycle or there are few reports on ribosome profiling under abiotic to infect different hosts, or the overlapping of templates stresses such are drought, varying external light conditions for transcription of mRNAs which, again, may be translated or in response to reactive oxygen species (Liu et al., 2013; during different steps of the infection cycle and/or in different Benina et al., 2015; Lei et al., 2015), but not under viral cellular environments. This brings us to various additional infection conditions, limiting the identification of potential methodological aspects that may require attention for further parallelisms. development of this research field: On the one hand, the Last but not least, the diversity of cis-acting translation dissection of the infection cycle is still a difficult task for elements identified in plant viruses may contribute to the design plant virologists, as there is a lack of experimental systems in of tools for synthetic biology (Ogawa et al., 2017), and in vectors which synchronous infections can be established. On the other for the overexpression of proteins in biofactory cell-free systems, hand, experimental systems appropriate for performing arrays cell cultures, or whole plants (Fan et al., 2012), or, perhaps, in

Frontiers in Plant Science | www.frontiersin.org 13 April 2017 | Volume 8 | Article 494 Miras et al. Non-canonical Translation in Plant RNA Viruses other organisms used for industrial overexpression of proteins, if MA supervised MM writing and wrote Sections Introduction mechanisms employed by plant viruses are universal or at least and Perspectives. conserved in the species of interest. ACKNOWLEDGMENTS AUTHOR CONTRIBUTIONS The research program in Aranda’s lab is supported by MM wrote Sections Intergenic Region Enhancers to grants AGL2015-65838 (MINECO, Spain) and ARIMNet2- Optimization of Viral mRNA Coding Capacity, WM, VT, EMERAMB(ERA-Net-618127, EU FP7). WM is funded by NIH and MA edited and added specific information to all sections, grant number R01 GM067104.

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